I. Field of the Invention
This invention relates to an assay element for determining the amount of a target substance contained in a sample fluid such as blood.
II. Description of the Related Art
Various immunoassay systems for quantifying a target substance such as various drugs, metabolites, hormones and vitamines, which is contained in a sample fluid such as blood have been developed. The principle of most of these systems is based on the immunoassay, especially enzyme immunoassay and multilayered assay films with high precision which offer simplified operations have been developed. These multilayered assay films comprise a transparent support, a reagent layer formed on the support and a porous spreading layer formed on the reagent layer. Various reagents which are necessary for the immunoassay are contained in the reagent layer. In operation, the sample fluid which may contain the target substance which is to be quantified is dropped on the spreading layer and the dropped fluid is uniformly spread in the spreading layer so that equal amount of the sample fluid per a unit area is supplied to the reagent layer. In the reagent layer, prescribed immunological reaction and the enzyme reaction are carried out. In the enzyme immunoassay, in most cases, coloring reaction is carried out by the aid of an enzyme, and the target substance is quantified by measuring the degree of the generation of color. With the multilayered assay film, this is conducted by impinging a light with a prescribed wavelength on the backside of the transparent support and then measuring the reflection density of the light. In a typical example of the conventional multilayered assay element, in the reagent layer, are contained a substance (antibody or antigen) which specifically reacts with the target substance to be quantified (antigen or antibody), the target substance labelled with a coenzyme such as FAD, glucose oxidase which is an apo enzyme which is activated by the coenzyme and glucose which is the substrate of the glucose oxidase, as well as peroxidase which generates color from hydrogen peroxide generated from glucose by the action of the glucose oxidase and 4-aminoantipyrine which is required for the coloring reaction, and the quantification of the target substance contained in the sample fluid is carried out by impinging a light with a specific wavelength on the backside of the transparent support and by measuring the reflection density of the light.
On the other hand, a bile pigment called bilirubin which has orange-yellow color is contained in the blood. Bilirubin is a normal metabolite which is produced mainly by the degradation of heme. The yellowness of the human serum is due to the existence of bilirubin and the bilirubin level is especially high in the blood of the patients suffering from porphyria. Since bilirubin is an orange-yellow pigment, if bilirubin is contained in the reagent layer of the above-mentioned multilayered assay element, the measurement of the generated color in the enzyme immunoassay is hindered. That is, when the wavelength of the color generated by the enzyme reaction is near the wavelength of the color of bilirubin, it is difficult to distinguish the color generated by the enzyme reaction from the color due to the existence of bilirubin, so that the precision of the immunoassay is degraded. Further, it is reported that bilirubin reacts with hydrogen peroxide, so that the precision of the enzyme immunoassay in which the generation of hydrogen peroxide is utilized is degraded. In particular, although blood can be analyzed without dilution with the recently developed dry chemistry type multilayered immunoassay elements, in this case, since the bilirubin level is high in the non-diluted blood, the degradation of the precision of the assay due to the existence of bilirubin is a serious problem. Especially, it is difficult to analyze the blood from the patients suffering from porphyria, in which bilirubin is contained at a high level, without diluting the blood. Thus, in such a case, the troublesome dilution operation is required.